Mathias Strupler

Second harmonic imaging and scoring of collagen in fibrotic tissues

We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals.

Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

Apports récents des techniques de quantification de la fibrose pour l’examen anatomopathologique en transplantation rénale

Chronic allograft injury can be diagnosed early at a pre-clinical stage by its histopathological changes. Interstitial fibrosis (IF), one of its main histopathological features, is currently assessed by semi-quantitative analysis according to the Banff classification. Subjective interpretation by the pathologist is the main limiting factor of such a method. Different morphometric approaches have been used to quantify IF. Point counting methods have been published but their use is very tedious. Thus, semi-automatic computerized measurements of IF in biopsy specimens immunostained for collagen III have been proposed. Most authors used Sirius Red-stained tissue examined under polarized or non-polarized light. However, morphometric methods are time-consuming. Automatic color segmentation image analysis is a new, rapid, and robust method to quantify IF on renal biopsies stained by light green trichrome. It can be recommended as being reliable and reproducible for routine use. Methodology based on second harmonic generation microscopy allows specific quantitative imaging of interstitial fibrosis with high reproducibility and may bring complementary informations in the future. The prognostic value of quantitative image analysis might be increased by techniques addressing the dynamics of fibrosis matrix generation.

Spectroscopic analysis of skin intrinsic signals for multiphoton microscopy

We recorded multiphoton images of human skin biopsies using endogenous sources of nonlinear optical signals. We detected simultaneously two-photon excited fluorescence (2PEF) from intrinsic fluorophores and second harmonic generation (SHG) from collagen. We observed SHG from fibrillar collagens in the dermis, whereas no SHG was detectable from the non fibrillar type IV collagen in the basal laminae. We compared these distinct behaviours of collagens I and IV in SHG microscopy to polarization-resolved surface SHG experiments on thin films of collagens I and IV molecules. We observed similar signals for both types of molecular films, except for the chiroptical contributions which are present only for collagen I and enhance the signal typically by a factor of 2. We concluded that SHG microscopy is a sensitive probe of the micrometer-scale structural organization of collagen in biological tissues. In order to elucidate the origin of the endogenous fluorescence signals, we recorded 2PEF spectra at various positions in the skin biopsies, and compared these data to in vitro spectroscopic analysis. In particular, we studied the keratin fluorescence and determined its 2PEF action cross section. We observed a good agreement between 2PEF spectra recorded in the keratinized upper layers of the epidermis and in a solution of purified keratin. Finally, to illustrate the capabilities of this technique, we recorded 2PEF/SHG images of skin biopsies obtained from patients of various ages.

Recent approaches of quantification of interstitial fibrosis in renal transplantation

Chronic allograft injury can be diagnosed early at a pre-clinical stage by its histopathological changes. Interstitial fibrosis (IF), one of its main histopathological features, is currently assessed by semi-quantitative analysis according to the Banff classification. Subjective interpretation by the pathologist is the main limiting factor of such a method. Different morphometric approaches have been used to quantify IF. Point counting methods have been published but their use is very tedious. Thus, semi-automatic computerized measurements of IF in biopsy specimens immunostained for collagen III have been proposed. Most authors used Sirius Red-stained tissue examined under polarized or non-polarized light. However, morphometric methods are time-consuming. Automatic color segmentation image analysis is a new, rapid, and robust method to quantify IF on renal biopsies stained by light green trichrome. It can be recommended as being reliable and reproducible for routine use. Methodology based on second harmonic generation microscopy allows specific quantitative imaging of interstitial fibrosis with high reproducibility and may bring complementary informations in the future. The prognostic value of quantitative image analysis might be increased by techniques addressing the dynamics of fibrosis matrix generation.

Apports récents des techniques de quantification de la fibrose pour l'examen anatomopathologique en transplantation rénale [Recent approaches of quantification of interstitial fibrosis in renal transplantation]

Chronic allograft injury can be diagnosed early at a pre-clinical stage by its histopathological changes. Interstitial fibrosis (IF), one of its main histopathological features, is currently assessed by semi-quantitative analysis according to the Banff classification. Subjective interpretation by the pathologist is the main limiting factor of such a method. Different morphometric approaches have been used to quantify IF. Point counting methods have been published but their use is very tedious. Thus, semi-automatic computerized measurements of IF in biopsy specimens immunostained for collagen III have been proposed. Most authors used Sirius Red-stained tissue examined under polarized or non-polarized light. However, morphometric methods are time-consuming. Automatic color segmentation image analysis is a new, rapid, and robust method to quantify IF on renal biopsies stained by light green trichrome. It can be recommended as being reliable and reproducible for routine use. Methodology based on second harmonic generation microscopy allows specific quantitative imaging of interstitial fibrosis with high reproducibility and may bring complementary informations in the future. The prognostic value of quantitative image analysis might be increased by techniques addressing the dynamics of fibrosis matrix generation.

Nonlinear microscopy of collagen fibers

We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.